Project Summary/Abstract Enzymes are well known for catalyzing chemical transformations with exquisite specificity and selectivity under environmentally benign conditions. Thus, there is a continuing need for the development of new enzymes that can effect critical synthetic transformations. One important type of transformations that is rarely present in current catalytic repertoire of biology is reactions for synthesis of organofluorine compounds. Organofluorine molecules have assumed a privileged position in modern pharmaceutical industry, which comprise ~ 30% of all pharmaceuticals and ~ 60% of all FDA-approved radiotracers for positron emission tomography (PET). The focus of this proposal is to generate new enzymatic platforms for the synthesis of organofluorine molecules. With protein engineering techniques like directed evolution, we will bring fluorination activities into existing proteins that share mechanistic features with synthetic fluorination reactions. These enzymatic platforms will expand the chemical space of biosynthesis tremendously and open up possibilities to develop whole new biosynthetic pathways for organofluorine synthesis. On their own, the enzymes developed would lead to highly efficient and selective synthetic routes to organofluorines that are currently unobtainable or feasible at scale. The structural and kinetic investigations of these new enzymes will greatly extend our understanding of enzymology and biochemistry to catalytic reactions unprecedented in nature. These new fluorination enzymes could be genetically incorporated into living hosts and coupled with existing biosynthetic pathways, enabling specific incorporation of fluorine groups into complex bioactive molecules. These research efforts will empower the development of new fluorine-based therapeutics and provide a paradigm for bringing non-biological chemistries to life.